Microbial pathogens can infect a host by one of several mechanisms. They may enter through a break in the integument induced by trauma, they may be introduced by vector transmission, or they may interact with a mucosal surface. The majority of human pathogens initiate disease by the last mechanism, i.e., following interaction with mucosal surfaces. Bacterial and viral pathogens that act through this mechanism first make contact with the mucosal surface where they may attach and then colonize, or be taken up by specialized absorptive cells (M cells) in the epithelium that overlay Peyer's patches and other lymphoid follicles [Bockman and Cooper, 1973, Am. J. Anat. 136:455-477; Owen et al., 1986, J. Infect. Dis. 153:1108-1118]. Organisms that enter the lymphoid tissues may be readily killed within the lymphoid follicles, thereby provoking a potentially protective immunological response as antigens are delivered to immune cells within the follicles (e.g., Vibrio cholerae). Alternatively, pathogenic organisms capable of surviving local defense mechanisms may spread from the follicles and subsequently cause local or systemic disease (i.e., Salmonella spp., poliovirus, rotavirus in immunocompromised hosts).
Secretory IgA (sIgA) antibodies directed against specific virulence determinants of infecting organisms play an important role in overall mucosal immunity [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor Laboratory, N.Y. p.p. 129-133]. In many cases, it is possible to prevent the initial infection of mucosal surfaces by stimulating production of mucosal sIgA levels directed against relevant virulence determinants of an infecting organism. Secretory IgA may prevent the initial interaction of the pathogen with the mucosal surface by blocking attachment and/or colonization, neutralizing surface acting toxins, or preventing invasion of the host cells. While extensive research has been conducted to determine the role of cell mediated immunity and serum antibody in protection against infectious agents, less is known about the regulation, induction, and secretion of sIgA. Parenterally administered inactivated whole-cell and whole-virus preparations are effective at eliciting protective serum IgG and delayed type hypersensitivity reactions against organisms that have a significant serum phase in their pathogenesis (i.e., Salmonella typhi, Hepatitis B). However, parenteral vaccines are not effective at eliciting mucosal sIgA responses and are ineffective against bacteria that interact with mucosal surfaces and do not invade (e.g., Vibrio cholerae). There is, however, recent evidence that parenterally administered vaccines may be effective against at least one virus, rotavirus, that interacts primarily with mucosal surfaces [Conner et al., 1993, J. Virol. 67:6633-6641]. Protection is presumed to result from transudation of antigen specific IgG onto mucosal surfaces for virus neutralization. Therefore, mechanisms that stimulate both serum and mucosal antibodies are important for effective vaccines.
Oral immunization can be effective for induction of specific sIgA responses if the antigens are presented to the T and B lymphocytes and accessory cells contained within the Peyer's patches where preferential IgA B-cell development is initiated. The Peyer's patches contain helper T (TH)-cells that mediate B-cell isotype switching directly from IgM cells to IgA B-cells. The patches also contain T-cells that initiate terminal B-cell differentiation. The primed B-cells then migrate to the mesenteric lymph nodes and undergo differentiation, enter the thoracic duct, then the general circulation, and subsequently seed all of the secretory tissues of the body, including the lamina propria of the gut and respiratory tract. IgA is then produced by the mature plasma cells, complexed with membrane-bound Secretory Component, and transported onto the mucosal surface where it is available to interact with invading pathogens [Strober and Jacobs, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 1-30; Tomasi and Plaut, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61]. The existence of this common mucosal immune system explains in part the potential of live oral vaccines and oral immunization for protection against pathogenic organisms that initiate infection by first interacting with mucosal surfaces.
A number of strategies have been developed for oral immunization, including the use of attenuated mutants of bacteria (i.e., Salmonella spp.) as carriers of heterologous antigens [Cardenas and Clements, 1992, Clin. Microbiol. Rev. 5:328-342; Clements et al., 1992, In: Recombinant DNA Vaccines: Rationale and Strategy, Isaacson (ed.), Marcel Decker, New York. p.p. 293-321; Clements and Cardenas, 1990, Res. Microbiol. 141:981-993; Clements and El-Morshidy, 1984, Infect. Immun. 46:564-569], encapsulation of antigens into microspheres composed of poly-DL-lactide-glycolide (PGL), protein-like polymers--proteinoids [Sanitago et al., 1993, Pharmaceutical Research 10:1243-1247], gelatin capsules, different formulations of liposomes [Alving et al., 1986, Vaccine 4:166-172; Garcon and Six, 1993, J. Immunol. 146:3697-3702; Gould-Fogerite and Mannino, 1993, In: Liposome Technology 2nd Edition. Vol. III, Gregoriadis (ed.)], adsorption onto nanoparticles, use of lipophilic immune stimulating complexes (ISCOMS) [Mowat and Donachie, 1991, Immunology Today 12:383-385], and addition of bacterial products with known adjuvant properties [Clements et al., 1988, Vaccine 6:269-277; Elson, 1989, Immunology Today 146:29-33; Lycke and Holmgren, 1986, Immunology 59:301-308; Lycke et al., 1992, Eur. J. Immunol. 22:2277-2281]. The two bacterial products with the greatest potential to function as oral adjuvants are cholera toxin (CT), produced by various strains of V. cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique.
The extensive diarrhea of cholera is the result of a potent exo-enterotoxin which causes the activation of adenylate cyclase and a subsequent increase in intracellular levels of cyclic 3-,5-adenosine monophosphate (cAMP). The cholera enterotoxin (CT) is an 84,000 dalton polymeric protein composed of two major, non-covalently associated, immunologically distinct regions or domains ("cholera-A" and "cholera-B") [Finkelstein and LoSpalluto, 1969, J. Exp. Med. 130:185-202]. Of these, the 56,000 dalton region, or choleragenoid, is responsible for binding of the toxin to the host cell membrane receptor, G.sub.M1 (galactosyl-N-acetylgalactosaminyl- (sialyl)-galactosyl-glucosyl ceramide), which is found on the surface of essentially all eukaryotic cells. Choleragenoid is composed of five non-covalently associated subunits, while the A region (27,000 daltons) is responsible for the diverse biological effects of the toxin.
The relationship of the two subunits of CT with respect to the immunologic properties of the molecule has been a source of considerable debate. On the one hand, CT is an excellent immunogen that provokes the development of both serum and mucosal antitoxin antibody responses when delivered orally. This finding is not new in that cholera patients are known to develop rises in titers of antitoxin antibodies during convalescence from clinical cholera [Finkelstein, 1975, Curr. Top. Microbiol. Immunol. 69:137-196]. One key finding of those investigating the nature of this response was the observation that CT, unlike most other protein antigens, does not induce oral tolerance against itself [Elson and Ealding, 1984, J. Immunol. 133:2892-2897; Elson and Ealding, 1984, J. Immunol. 132:2736-2741]. This was also found to be true when just the B-subunit was fed to mice, an observation substantiated by the cholera vaccine field trials in Bangladesh in which oral immunization with B-subunit combined with killed whole cells gave rise to mucosal as well as systemic antitoxin antibody responses [Svennerholm et al., 1984 J. Infect. Dis. 149:884-893].
In addition to being a potent oral immunogen, CT has a number of other reported immunologic properties. As indicated above, Elson and Ealding [Elson and Ealding, 1984, J. Immunol. 133:2892-2897] observed that orally administered CT does not induce tolerance against itself. Moreover, simultaneous oral administration of CT with a soluble protein antigen, keyhole limpet hemocyanin (KLH), resulted in the development of secretory IgA responses against both CT and KLH and also abrogated the induction of oral tolerance against KLH. These findings were subsequently confirmed and extended by Lycke and Holmgren [Lycke and Holmgren, 1986, Immunology 59:301-308]. The confusion arises when one attempts to define the role of the A and B subunits of CT with respect to the adjuvant properties of the molecule. The following observations, as summarized by Elson [Elson, 1989, Immunology Today 146:29-33], are the basis for that confusion:
CT does not induce oral tolerance against itself [Elson and Ealding, 1984, J. Immunol. 133:2892-2897]. PA1 CT-B does not induce oral tolerance against itself [Elson and Ealding, 1984, J. Immunol. 133:2892-2897]. PA1 CT can prevent the induction of tolerance against other antigens with which it is simultaneously delivered and also serve as an adjuvant for those antigens [Elson and Ealding, 1984, J. Immunol. 133:2892-2897; Lycke and Holmgren, 1986, Immunology 59:301-308]. PA1 CT can act as an adjuvant for CT-B [Elson and Ealding, 1984, J. Immunol. 133:2892-2897]. PA1 Heat aggregated CT has little toxicity but is a potent oral immunogen [Pierce et al., 1983, Infect. Immun. 40:1112-1118]. PA1 CT-B can serve as an immunologic "carrier" in a traditional hapten-carrier configuration [Cebra at al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133; McKenzie and Halsey, 1984, J. Immunol. 133:1818-1824]. PA1 1. Simultaneous administration of LT with OVA was shown to prevent the induction of tolerance to OVA and to increase the serum anti-OVA IgG response 30 to 90 fold over OVA primed and PBS primed animals, respectively. This effect was determined to be a function of the enzymatically active A-subunit of the toxin since the B-subunit alone was unable to influence tolerance induction. PA1 2. Animals fed LT with OVA after an initial OVA prime developed a significantly lower serum IgG and mucosal IgA anti-OVA response than those fed LT with OVA in the initial immunization, indicating that prior exposure to the antigen reduces the effectiveness of LT to influence tolerance and its ability to act as an adjuvant. LT was not able to abrogate tolerance once it had been established. This was also found to be true for CT when animals were pre-immunized with OVA prior to oral ovalbumin plus CT and offers some insight into the beneficial observation that antibody responses to nontarget dietary antigens are not increased when these adjuvants are used. PA1 3. Serum IgG and mucosal IgA responses in animals receiving LT on only a single occasion, that being upon first exposure to antigen, were equivalent to responses after three OVA/LT primes, indicating that commitment to responsiveness occurs early and upon first exposure to antigen. It was also demonstrated that the direction of the response to either predominantly serum IgG or mucosal IgA can be controlled by whether or not a parenteral booster dose is administered. PA1 4. Simultaneous administration of LT with two soluble protein antigens results in development of serum and mucosal antibodies against both antigens if the animal has no prior immunologic experience with either. This was an important finding since one possible application of LT as an adjuvant would be for the development of mucosal antibodies against complex antigens, such as killed bacteria or viruses, where the ability to respond to multiple antigens would be important.
A number of researchers have concluded from these findings that the B-subunit must possess some inherent adjuvant activity. The findings of Cebra et al. [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133], Lycke and Holmgren [Lycke and Holmgren, 1986, Immunology 59:301-308], and Liang et al. [Liang et al., 1988, J. Immunol. 141:1495-1501] would argue against that conclusion. Cebra et al. [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133] demonstrated that purified CT-B was effective at raising the frequency of specific anti-cholera toxin B-cells in Peyer's patches when given intraduodenally but, in contrast to CT, did not result in significant numbers of IgA committed B-cells. Lycke and Holmgren [Lycke and Holmgren, 1986, Immunology 59:301-308] compared CT and CT-B for the ability to enhance the gut mucosal immune response to KLH by measuring immunoglobulin secreting cells in the lamina propria of orally immunized mice. They found no increase in anti-KLH producing cells in response to any dose of B-subunit tested in their system. Finally, Liang et al. [Liang et al., 1988, J. Immunol. 141:1495-1501] found no adjuvant effect when CT-B was administered orally in conjunction with inactivated Sendai virus.
Where adjuvant activity has been observed for isolated B-subunit, it has typically been for one of two reasons. First, a traditional method of preparing B-subunit has been to subject holotoxin to dissociation chromatography by gel filtration in the presence of a dissociating agent (i.e., guanidine HCl or formic acid). The isolated subunits are then pooled and the dissociating agent removed. B-subunit prepared by this technique is invariably contaminated with trace amounts of A-subunit such that upon renaturation a small amount of holotoxin is reconstituted. The second reason has to do with the definition of an immunologic carrier. Like many other soluble proteins, B-subunit can serve as an immunologic vehicle for presentation of antigens to the immune system. If those antigens are sufficiently small as to be poorly immunogenic, they can be made immunogenic in a traditional hapten-carrier configuration. Likewise, there is a "theoretical" immune enhancement associated with B-subunit, especially for oral presentation, in that B-subunit binds to the surface of epithelial cells and may immobilize an attached antigen for processing by the gut associated lymphoid tissues. However, any potential advantage to this mechanism of antigen stabilization may be offset by the distribution of the antigen across non-immunologically relevant tissues, i.e., the surface of intestinal epithelial cells. In context of the mucosal responsiveness, the immunologically relevant sites are the Peyer's patches, especially for antigen-specific T cell-dependent B cell activation [Strober and Jacobs, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 1-30, Tomasi and Plaut, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61; Brandtzaeg, 1989, Curr. Top. Microbiol. Immunol. 146:13-25]. Thus, the events up to isotype switching from IgM cells to IgA B-cells occurs in the Peyer's patches. Antigens localized on the epithelial cell surface may contribute to antigen induced B cell proliferation in that the class II positive villous epithelial cells may act as antigen presenting cells for T cell activation at the secretory site, thereby increasing cytokine production, terminal B cell differentiation, increased expression of secretory component, and increased external transport of antigen specific IgA [Tomasi, T. B., and A. G. Plaut. 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61]. The relationships of these events have not been clearly defined for B-subunit as a carrier of other antigens and use of the term "adjuvant" would seem inappropriate for such an effect.
It is clear that the adjuvant property of the molecule resides in the holotoxin in which B-subunit is required for receptor recognition and to facilitate penetration of the A-subunit into the cell. The A-subunit is also required for adjuvant activity, presumably as a function of its ADP-ribosylating enzymatic activity and ability to increase intracellular levels of cAMP (see below). The B-subunit alone may act as a carrier of other antigens in that when conjugated to those antigens they can be immobilized for processing by the gut associated lymphoid tissues.
Although LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique, including a 20% difference in nucleotide and amino acid sequence homology [Dallas and Falkow, 1980, Nature 288:499-501]. The two toxins have the same subunit number and arrangement, same biological mechanism of action, and the same specific activity in many in vitro assays [Clements and Finkelstein, 1979, Infect. Immun. 24:760-769; Clements et al., 1980, Infect. Immun. 24:91-97].
There are, however, significant differences between these molecules that influence not only their enterotoxic properties, but also their ability to function as adjuvants. To begin with, unlike CT produced by V. cholerae, LT remains cell associated and is only released from E. coli during cell lysis [Clements and Finkelstein, 1979, Infect. Immun. 24:760-769]. CT is secreted from the vibrio as soon as it is synthesized and can be readily identified in, and purified from, culture supernatants. Consequently, in contrast to CT, LT is not fully biologically active when first isolated from the cell. Consistent with the A-B model for bacterial toxins, LT requires proteolysis and disulfide reduction to be fully active. In the absence of proteolytic processing, the enzymatically active A.sub.1 moiety is unable to dissociate from the A.sub.2 component and cannot reach its target substrate (adenylate cyclase) on the basolateral surface of the intestinal epithelial cell. This is also true for CT, but proteases in the culture supernatant, to which the toxin is exposed during purification, perform the proteolysis. Since LT is not fully biologically active, it is difficult to identify during purification using in vitro biological assays such as the Y-1 adrenal cell assay or permeability factor assay.
This difference in activation of the isolated material results in differences in response thresholds for LT and CT in biologic systems. For instance, CT induces detectable net fluid secretion in the mouse intestine at a dose of 5-10 .mu.g. LT induces detectable net secretion in the mouse intestine at levels above 100 .mu.g. In the rabbit ligated ileal loop, the difference is dramatic and clear cut. Moreover, in primates LT has been shown not to induce fluid secretion at any dose tested up to 1 milligram. This is 200 times the amount of CT reported to induce positive fluid movement in humans. When LT is exposed to proteolytic enzymes with trypsin-like specificity, the molecule becomes indistinguishable from CT in any biologic assay system. This was demonstrated clearly by Clements and Finkelstein [Clements and Finkelstein, 1979, Infect. Immun. 24:760-769].
In addition to the above reported differences, LT has an unusual affinity for carbohydrate containing matrices. Specifically, LT, with a molecular weight of 90,000, elutes from Sephadex columns (glucose) with an apparent molecular weight of 45,000 and from Agarose columns (galactose) with an apparent molecular weight of 0. That is, it binds to galactose containing matrices and can be eluted from those matrices in pure form by application of galactose. LT binds not only to agarose in columns used for purification, but more importantly, to other biological molecules containing galactose, including glycoproteins and lipopolysaccharides. This lectin-like binding property of LT results in a broader receptor distribution on mammalian cells for LT than for CT which binds only to G.sub.M1. This may account in part for the reported differences in the abilities of these two molecules to induce different helper T lymphocyte responses [McGhee et al., 1994, Mucosal Immunology Update, Spring 1994, Raven Press, New York. p. 21].
In these studies reported by McGhee et al. [McGhee et al., 1994, Mucosal Immunology Update, Spring 1994, Raven Press, New York. p. 21], it was shown that oral immunization of mice with vaccines such as tetanus toxoid (TT) with CT as a mucosal adjuvant selectively induces T.sub.H 2 type cells in Peyer's patches and spleens as manifested by TH cells which produce IL-4 and IL-5, but not IL-2 or INF-gamma. [For a more complete review of the cytokine network see Arai et al., 1990, Ann. Rev. Biochem. 59:783-836]. Importantly, when CT was used as a mucosal adjuvant it also enhanced antigen-specific IgE responses in addition to the IgA response. Such enhancement of IgE responses seriously compromises the safety of CT as a mucosal adjuvant due to the prospect of inducing immediate-type hypersensitivity reactions. In contrast, LT induces both T.sub.H 1 and T.sub.H 2 cells and predominantly antigen-specific IgA responses without IgE responses when used as an orally administered mucosal adjuvant.
The two molecules also have many immunologic differences, as demonstrated by immunodiffusion studies [Clements and Finkelstein, 1978, Infect. Immun. 21:1036-1039; Clements and Finkelstein, 1978, Infect. Immun. 22:709-713], in vitro neutralization studies, and the partial protection against LT associated E. coli diarrhea in volunteers receiving B-subunit whole cell cholera vaccine [Clemens et al., 1988, J. Infect. Dis. 158:372-377].
Taken together, these findings demonstrate that LT and CT are unique molecules, despite their apparent similarities, and that LT is a practical oral adjuvant while CT is not.
The demonstration of the adjuvant properties of LT grew out of an investigation of the influence of LT on the development of tolerance to orally administered antigens by one of the present inventors. It was not clear whether or not LT would also influence the induction of oral tolerance or exhibit the adjuvant effects demonstrated for CT, given the observed differences between the two molecules. Consequently, the present inventors examined a number of parameters, including the effect of LT on oral tolerance to OVA and the role of the two subunits of LT in the observed response, the effect of varying the timing and route of delivery of LT, the effect of prior exposure to OVA on the ability of LT to influence tolerance to OVA, the use of LT as an adjuvant with two unrelated antigens, and the effect of route of immunization on anti-OVA responses. The results obtained from these studies [Clements et al., 1988, Vaccine 6:269-277; Clements et al., 1988, Abstract No. B91, 88th Ann. Meet. Am. Soc. Microbiol.] are summarized below:
Studies by Tamura et al., [Tamura et al., U.S. Pat. No. 5,182,109] demonstrated that LT and/or CT administered intranasally enhanced the antibody titer against a co-administered antigen. However, nowhere in Tamura et al. is it taught that these toxins can induce a protective immune response when administered orally.
Clearly, LT has significant immunoregulatory potential, both as a means of preventing the induction of tolerance to specific antigens and as an adjuvant for orally administered antigens and it elicits the production of both serum IgG and mucosal IgA against antigens with which it is delivered. This raises the possibility of an effective immunization program against a variety of pathogens involving the oral administration of killed or attenuated agents or relevant virulence determinants of specific agents. However, the fact that this "toxin" can stimulate a net lumenal secretory response when proteolytically cleaved, as by gut proteases, or when administered in high enough concentrations orally, may hinder investigation into its potential or prevent its use under appropriate conditions. This problem could be resolved if LT could be "detoxified" without diminishing the adjuvant properties of the molecules. In order to appreciate how this might be accomplished, it is necessary to further analyze the mechanism of action of the LT and CT and the structural and functional relationships of these molecules. As indicated previously, both LT and CT are synthesized as multisubunit toxins with A and B components. After the initial interaction of the toxin with the host cell membrane receptor, the B region facilitates the penetration of the A-subunit through the cell membrane. On thiol reduction, this A component dissociates into two smaller polypeptide chains. One of these, the A.sub.1 piece, catalyzes the ADP-ribosylation of the stimulatory GTP-binding protein (G.sub.S) in the adenylate cyclase enzyme complex on the basolateral surface of the epithelial cell and this results in increasing intracellular levels of cAMP. The resulting increase in cAMP causes secretion of water and electrolytes into the small intestine through interaction with two cAMP-sensitive ion transport mechanisms involving 1) NaCl co-transport across the brush border of villous epithelial cells, and 2) electrogenic Na.sup.+ dependent Cl.sup.- secretion by crypt cells [Field, 1980, In: Secretory diarrhea, Field et al. (ed.), Waverly Press, Baltimore. p.21-30]. The A subunit is also the principal moiety associated with immune enhancement by these toxins. This subunit then becomes a likely target for manipulation in order to dissociate the toxic and immunologic functions of the molecules. A recent report by Lycke et al. [Lycke et al., 1992, Eur. J. Immunol. 22:2277-2281] makes it clear that alterations that affect the ADP-ribosylating enzymatic activity of the toxin and alter the ability to increase intracellular levels of cAMP also prevent the molecule from functioning as an adjuvant. Consequently, another approach to detoxification must be explored.